Acute myeloid leukemia (AML) is a genetically heterogeneous bone marrow cancer affecting at least 15,000 individuals per year in the US and causing significant morbidity and mortality. We identified BCOR loss of function mutations among a set of genes that is specifically mutated in AML that arises after myelodysplastic syndromes, is common among older patients, and is associated with poor clinical outcomes. The mechanisms by which BCOR mutations drive leukemogenesis are not understood. In preliminary studies, we showed that Bcor mutations promote increased hematopoietic stem cell self-renewal and sensitize primary cells to activating RAS mutations. These represent the first reported mouse models of Bcor-deficient myeloid disease. We also demonstrate for the first time that BCOR protein interacts in human AML cell lines with components of a variant polycomb repressive complex 1 (PRC1), suggesting a functional importance of ubiquinated histone 2A lysine 119 (H2AK119ub) in AML development. Our hypothesis is that somatic mutations affecting BCOR cause failure to recruit PRC2 to polycomb repressive targets via reduction of H2AK119ub. To define the molecular basis of BCOR function during initiation and progression of myeloid malignancies, we propose the following Specific Aims 1) Define the contribution of Bcor deletion to myeloid transformation in primary Tet2-/- hematopoietic cells. We will use CRISPR/Cas9 gene editing in primary mouse Tet2-/- cells in vitro and in vivo to define the functional impact of Bcor loss of function mutations on hematopoietic stem cell self-renewal and lineage-specific differentiation. 2) Identify the role of RAS mutations in promoting transformation of Bcor- deficient cells. We will take advantage of novel in vitro and in vivo models to identify the cooperative effects of activating RAS mutations during transformation of Bcor-deficient cells. 3) Define the impact of BCOR deficiency on histone epigenetic modifications during myeloid leukemogenesis. We will define the global and locus-specific effects of BCOR deletion on histone H3 epigenetic marks in a human AML cell line and assess whether genetic and chemical targeting of EZH2 attenuates BCOR-associated transformation. Collectively, the information learned in these studies will provide new insights into the biology o AML and lay the groundwork for developing novel targeted therapies. The applicant, Dr. Coleman Lindsley, has outlined a five-year career development plan to meet his goal of becoming an independent investigator in translational leukemia research. Dr. Lindsley has assembled an Advisory Committee of internationally recognized experts to provide scientific and career mentorship. He has established collaborations with experts in cancer epigenetics, leukemia genetics, and applied biostatistics to provide experimental advice and specific training in the field. Dana-Farber Cancer Institute is an optimal environment for completion of Dr. Lindsley's scientific and career goals, given its outstanding research community and long-standing record for training independent physician-scientist.